A huge amount of chemical substances are processed in many fields, including those related to chemicals, pharmaceuticals, biology and medicine. In such fields, there is frequently a requirement that a target analyte of interest has to be separated from a mixture. For example, in fields such as chemical and pharmaceutical industries, chemical analytes are synthesized, and chemical synthesis tends to generate reaction mixtures that contain target analytes of interest as well as other species such as (potentially multiple) by-products and unreacted reactants. As a further example, in biological, medical, food and other industries, mixtures that are readily obtained contain various target analytes of interest and other species.
Frequently, it is desired to detect and/or characterize (e.g. determine a concentration of) a target analyte of interest; e.g. detect and determine a concentration of bacteria in food, measure a concentration of glucose in blood, etc. Many other examples are readily apparent to those skilled in the art. It is quite common to detect and/or characterize target analytes of interest directly from mixtures. It is also quite common to subject mixtures to separation processes whereby target analytes of interest are obtained with increased purity to aid in their detection, characterization and further use.
Separation and/or detection methods include, but are not restricted to, thin film chromatography, flash column chromatography, high performance liquid chromatography (HPLC) and electrophoresis. Separation methods may be analytical in nature to characterize mixtures or preparatory in nature to generate separations in quantitative yields. To separate a target analyte of interest from a mixture, separation methods pass mixtures through various materials and exploit a fact that different species and target analytes of interest pass through the materials at different rates.
For example, in liquid chromatography, mixtures are passed through various packing materials in a column. Target analytes of interest and various other species travel through separatory materials at different rates, depending on various factors such as different interactions experienced by various species or target analytes of interest arising from a nature of solvents, chemical nature of packing materials, an existence and size of pores in the packing materials, etc. In a successful separation process, target analytes of interest and various other species in the mixtures exit the column at different times, and can thus be separated. If a target analyte of interest has a distinct visible colour, the time when the target analyte of interest exits a separation apparatus can be determined visually. If the target analyte of interest does not have a distinct visible colour, however, other physical properties of the target analyte of interest have to be measured to aid in its selection from other species in the mixture. Methods and devices that can yield quantitative measures for a degree of separation of target analytes of interest from other species are highly desirable.
Optical measurements are most frequently used for such purposes. For instance, surface plasmon resonance (SPR) is used to monitor binding of target analytes of interest to surfaces and thereby detect a presence of such target analytes. In chromatography and electrophoresis, optical spectroscopy using ultra-violet (UV) and/or visible light is frequently employed to obtain absorption spectra of target analytes of interest and other species and thereby monitor their separation. Depending on chemical natures and structures of target analytes of interest and other species, their absorption strength may be different. Apparatus for optical measurements can include UV lamps, lasers, lenses, detectors, and other optical elements and tend to be large and relatively expensive. Also, often target analytes of interest and other species do not have UV or visible absorption features that are suitable and optical methods cannot be utilized. Further, many biological target analytes of interest, for example proteins, are frequently obtained only in small quantities so that very high detection sensitivities and high-signal-to-noise ratio are required. To overcome these challenges, optical methods and devices rely on labels, for example, fluorescent labels. However, this requires modifying the target analyte of interest, which is usually undesirable, and requires significant labour, time and, as a result, expense. Optical devices and methods that rely on measurements of bulk index of refraction tend to be sensitive to temperature and pressure of solvents. For example, mechanical deformations induced by temperature and pressure changes result in changes in signal that compete with changes in signal induced by target analytes of interest. Also, when performing chromatography such as HPLC, for example, it is frequently desirable to use a gradient elution, that is, to use mixtures of solvents containing two or more components and to vary fractions of components present in the mixture systematically as the separation proceeds. Since the index of refraction of the solvent varies significantly as the fractions of components vary, it is difficult to detect small changes in index of refraction induced by small amounts of target analytes of interest. As a result, index of refraction measurements are not used with gradient elution.
A number of inventions are directed towards detecting a presence of target analytes of interest based on changes in electrical resistance (or equivalently resistivity, conductance or conductivity) of a circuit. U.S. Pat. No. 6,824,974 B2 teaches detection of a target analyte of interest using a biomolecule that spans a gap between two electrodes. Binding of a target analyte of interest changes conductivity between the two electrodes.
U.S. Pat. No. 6,458,327 B1 teaches an electronic device, especially a chemical sensor, comprising a nanoparticle structure configured such that a current path is defined through said nanoparticle structure and analyte molecules change the conductivity of the structure.
U.S. Pat. No. 5,194,133 discloses a sensor device for the analysis of a sample fluid comprising an elongated channel, a material in the channel causing separation of a sample fluid, enzymes, and pairs of sensing electrodes along the walls of the channel. Enzymes in the channel react with enzyme substrates in the sample fluid, changing conductivity of the sample fluid and thereby signalling a presence of the enzyme substrates.
U.S. Pat. No. 4,920,047 describes a method and apparatus for determining the presence of, the concentration of, or the absence of, immunologically active substances in liquid media by measuring any change of electrical impedance of an electrode. The electrode is provided with immunologically active substances, such as antigens or antibodies, which in turn provide binding sites for complementary immunologically active substances, such as antibodies or antigens, respectively. If the electrode is exposed to complementary immunologically active substances, binding sites become unavailable; otherwise, the binding sites remain available. The electrode is subsequently exposed to an enzyme that is also capable of binding to the binding sites and capable of generating an insoluble reaction product. The insoluble reaction product can deposit and adhere to the electrode thereby changing its impedance and indirectly signalling the presence of, the concentration of, or the absence of, complementary immunologically active substances in liquid media, such as water or saline.
Resistance-based methods and devices are limited by a competition between influences of target analytes of interest vs. those of media such as water, and the like which are conducting. As a result, sensitivity can be limited in devices and methods that require operation in such media and that attempt to detect target analytes of interest directly. To overcome such difficulties, devices and methods may employ amplification of sensitivity (e.g. through use of enzymes to generate significant product to signal detection) or removal of devices from such media; however, these approaches require additional steps and, therefore, resources such as time, expense, etc. Also, in chromatography applications such as HPLC, it is frequently desired to detect target analytes of interest that are non-conducting and that are dissolved in non-conducting media.
A number of inventions, therefore, have also been directed towards detecting a presence of target analytes of interest based on changes in electrical capacitance, C. Such inventions utilize a principle that capacitance is proportional to the dielectric constant of a medium in a region sensed by electric fields of the capacitor. If the region contains a mixture of two media, A and B, with respective dielectric constants ∈A and ∈B, then the capacitance is proportional to an effective dielectric constant, ∈, which is a function of ∈A, ∈B and volume fractions of A and B. For a large parallel plate capacitor, approximately C=∈ A/d, where A is area and d is separation of the parallel plates. The example of a parallel plate capacitor is used for illustrative purposes only and is not intended to limit the scope of this invention. The above formula for capacitance for a parallel plate capacitor assumes that the electric field is localized in the volume A·d between the parallel plates. In practise, for finite size plates, there is a fringe electric field that extends beyond edges of the parallel plates to length scales that are on the order of d; nevertheless, C is still proportional to ∈. The impedance, ZC, of a capacitor at a frequency, ω, is ZC=(j ω C)−1. When the capacitor is driven by a time dependent voltage, V, the voltage generates a time dependent electric field which senses ∈ in a region. Depending on ∈ sensed by the electric field, the electric field induces a polarization in the region, which in turn induces a time dependent charge on the capacitor. The resulting capacitative current, I, is I=V/ZC=jωCV. To facilitate measurement, the capacitative current is typically amplified, by an amount RG, generating a measured voltage, VG=jωRGCV. For a parallel plate capacitor, VG=jωRG∈A V/d; thus, the measured voltage across the capacitor is proportional to ∈.
Accordingly, in capacitance-based methods and devices, a change in E generates a change in VG. Such a change in ∈ occurs, for example, when a target analyte of interest with a first dielectric constant enters the region sensed by the electric field and displaces media in the region with a second effective dielectric constant of a different value. If the target analyte of interest has a small dielectric constant and the media includes solvents such as water, saline, electrolytes, and the like, which have large effective dielectric constants by virtue of their non-insulating nature, large changes in ∈ can be realized. If a target analyte of interest is located in the region sensed by the electric field, and an object (for example a conducting bead) with a large effective dielectric constant is attached to the target analyte of interest, thereby displacing media with smaller effective dielectric constant, again large changes in ∈ can be generated. Such large changes in ∈ have been exploited in a number of devices and methods designed to detect target analytes of interest.
U.S. Pat. No. 6,764,583 B2 teaches impedance measurements between electrodes in an electric field to detect the presence of pathogens trapped in the electric field. The pathogens change the impedance between electrodes by changing the dielectric material between the electrodes. Subsequently in U.S. Pat. No. 6,846,639 B2, Miles et al. teach using beads coated with antibodies to aid in the detection of pathogens. The beads stick to pathogens trapped in the electric field, producing an additional change in the impedance.
United States Patent Publication No. 2005/0227373 A1 discloses a method and device for high sensitivity detection of the presence of DNA and other probes. A presence of a target sample on a substrate is capacitatively detected by binding the target sample to selective binding sites on the substrate, the target sample being directly or indirectly labelled with conducting labels, and capacitatively detecting the presence of the conductive labels.
United States Patent Publication No. 2002/0192653 A1 is directed towards impedance-based chemical and biological imaging sensor apparatus and methods. The imaging sensor consists of a two-dimensional array of impedance electrode elements separated from chemical or biological samples contained in fluids by a fluid-impervious layer. Changes in capacitance due to impedance changes at an outer surface of the fluid-impervious layer are detected during interrogation of electrode elements. The imaging chip does not respond to dry pollen, but if the particles are suspended in dilute phosphate buffer and a trace surfactant, the particles can be imaged in contrast.
U.S. Pat. No. 5,846,708 teaches a method and apparatus for identifying molecular structures within a sample substrate using a monolithic array of test sites. In an electrical embodiment of the invention, a substance having a molecular structure is applied to the test sites, each test site having a probe capable of binding (hybridizing) with a known molecular structure. Hybridized molecules can be detected, in accordance with one embodiment of the invention, by sensing the change in the dissipation of a capacitor formed at the test site. At the resonance frequency of a DNA molecule in aqueous solution, the imaginary part of ∈ can be approximately 10 to 100 times larger than its value for an aqueous solution without the DNA. The patent teaches that an LCR meter may be used to measure the resistance.
U.S. Pat. No. 5,187,096 discloses an apparatus and method for monitoring cell-substrate impedance using an array of electrode pairs. Each electrode pair includes a large counter electrode and a small active electrode. An AC current is applied between electrodes of each pair, while the voltage is monitored using a phase sensitive detector. Cells are cultured on the small electrodes. As the cells attach and flatten out on the electrode surface, they cause large changes in electrical impedance of the system.
United States Patent Publication No. 2006/0216203 is directed to a multi-well sample module having integrated impedance measuring electrodes which allow for the generation of an electric field within each well and the measuring of a change in impedance of each of the wells contents. The electric field generated by the electrodes extend from the electrodes roughly to the gap between the electrodes. Cells experience this electric field. Measurement of the total current allows calculation of the cell impedance from the impedance measurement. The impedance measurement is performed by measuring the current resulting from an applied alternating voltage. Both the magnitude and phase are part of the impedance.
U.S. Pat. No. 4,822,566 discloses an apparatus for detecting the presence and/or measuring the concentration of an analyte in a fluid medium. The apparatus relies on biospecific binding between a biochemical binding system and the analyte to change the dielectric properties of a capacitative affinity sensor. The biological affinity sensor is optimized by: (1) adjusting the thickness and dielectric properties of a passivation layer to generally match the impedance of the biological binding system; and (2) minimizing the double layer capacitance (of the non-insulating fluid system) in order to maximize capacitance changes associated with the biological binding system.
It is desirable to have a general method and device that are label-free and capable of measuring even small changes in effective dielectric constant. As an example, in chromatographic separations, target analytes of interest may be insulating and dissolved in solvents such as alkanes or benzene, which are also insulating. In such cases, differences between effective dielectric constants of solvents and of mixtures of solvents and target analytes of interest are small. To generate sufficiently large changes in measured voltages even for such applications, using a parallel plate capacitor geometry as an example, it is desirable to chose advantageously RG, ω, A, d, and V. Increasing V and RG results in larger changes in measured voltages. Increasing ω also results in larger changes in measured voltage. Measurement of voltages at various ω and regression analysis of such measurements results in increased accuracy in determination of changes in ∈. A and d (and in general for non-parallel plate capacitors, the volume sensed by the electric field) can be engineered to optimize changes in measured voltages. If the volume sensed by the electric fields is too small, then only a small portion of space occupied by target analytes of interest will be sensed, which is undesirable. If the volume is too large, then the change in ∈ induced by the target analytes of interest will be small, which, too, is undesirable. An intermediate choice is preferred. In one optical embodiment of the present invention, near field optics principles can be used to confine electric fields to much smaller volumes than in far field optics. By engineering sizes, shapes, spacings, orientation, etc of electrodes it is possible to engineer electric fields in electronics. Therefore, it will be apparent to those skilled in the arts that optimization of the electric fields for a given target analyte of interest is possible and desirable to detect changes in ∈, not just in electronics but in electromagnetics generally.
In order to increase changes in VG induced by changes in ∈, in turn induced by a target analyte of interest, it is desirable to maximize the fraction of volume occupied by the target analyte of interest in the region sensed by the electric field. This can be accomplished by engineering electric fields as disclosed above and further by incorporating into the region sensed by the electric field, atomic species, functional groups, molecules, and more generally chemical and/or biological discrimination elements that interact with target analytes of interest. For example, if the region sensed by the electric field is at or near a surface and the target analyte of interest is a strand of DNA, then functionalizing the surface with a complementary strand of DNA can generate higher concentrations of the strand of DNA near the surface than in solution. Many interactions can be exploited in such a fashion to increase concentrations of target analytes of interest and will be apparent to those skilled in the arts. The interactions include electromagnetic and/or quantum interactions such as those that give rise to antigen-antibody paring, DNA hybridization, and interactions between other biological species, various chemical phenomena such as bonding, solubility, and the like. Such interactions generate various degrees of chemical and/or biological discrimination and will be apparent to those skilled in the arts. Such increases of concentrations have an advantageous feature of overcoming a problem that arises generally for devices and methods that rely on measurements of bulk properties such as conductivity and bulk dielectric constant. As solvent composition changes during gradient elution, there arises a large change in bulk properties, making detection of small changes generated by target analytes of interest difficult. Increasing the volume fraction of target analytes of interest in the region sensed by the electric field has advantageous effects of reducing the volume fraction of the solvents and mitigating the detrimental influence of changing solvent composition.
If media in which target analytes of interest are dissolved are non insulating, the media will have finite conductance and therefore resistance. Hence they generate dissipation in the capacitance, a real component in the measured voltage, a complex component in ∈ and a complex component in the measured voltage that involves both resistance and capacitance. As resistance decreases, current increases, leading to saturation of electronics especially if RG is increased to detect small changes in ∈. Determination of capacitative impedance, and therefore, small changes in ∈ become difficult. To address this problem, it is desirable to develop methods and devices employing an insulating region that impedes external current flow so as to permit detection of even small changes in ∈.
A simple-to-use, inexpensive, label-free, portable, quantitative, robust, sensitive, structurally and chemically stable and generally applicable invention for detecting, distinguishing, and characterizing target analytes of interest and other species is, therefore, highly desirable. In particular, it would be highly desirable to have an invention that is based on a property universally possessed by all target analytes of interest (for example, ∈) and that is insensitive to changes other than those induced by target analytes of interest. Such an invention would have many other applications, besides monitoring separation of mixtures. These applications include, but are not restricted to, monitoring interactions between surfaces functionalized with chemical and/or biological discrimination elements (such as unfunctionalized molecules, mono-functionalized molecules, bi-functionalized molecules, poly-functionalized molecules, oligomers, polymers, catalysts, cells, bacteria, viruses, enzymes, proteins, heptans, saccharides, lipids, glycogens, enzyme inhibitors, enzyme substrates, neurotransmitters, hormones, antigens, antibodies, DNA, and/or RNA), and pharmaceutical, biological and/or medically related compounds (such as drugs, DNA, RNA, proteins, antigens, antibodies, heptans, saccharides, lipids, glycogens, enzyme inhibitors, enzyme substrates, neurotransmitters, hormones, viruses, bacteria, cells, etc.) The invention can also be used for quality control tests in which results obtained using a control system are compared with those obtained using a test system. Such tests would be useful to monitor whether a chemical has become contaminated for instance. The invention can further be used in tests for monitoring water. Other uses for such an invention will be apparent to those skilled in the arts.